Objectives: To investigate the inhibitory effect of GSK484, a PAD4 inhibitor, on H3Cit expression following sepsis and its effects for improving sepsis-induced endothelial dysfunction.
Methods: Eighteen C57BL/6 mice were randomized into sham-operated group, sepsis model group and GSK484 treatment group (n=6), and in the latter two groups, models of sepsis were established by cecal ligation and puncture (CLP). The mice in GSK484 treatment group were given an intraperitoneal injection of GSK484 (4 mg/kg) on the second day following the surgery. Twenty-four hours after the injection, the mice were euthanized for measurement of serum levels of VEGF, ESM-1, IL-6 and IL-1β using ELISA. Lung tissue pathology was observed with HE staining, and pulmonary expressions of F-actin, VE-cadherin, ZO-1 and H3Cit proteins were detected using immunofluorescence staining and Western blotting. In primary cultured of mouse lung microvascular endothelial cells, the effect of stimulation with LPS (10 μg/mL) for 24 h on tube formation, proliferation, apoptosis and expressions of VEGF, ESM-1, IL-6 and IL-1β were assessed using CCK-8 assay, flow cytometry and ELISA.
Results: Compared to the sham-operated mice, the septic mice exhibited significant lung tissue pathologies characterized by vascular congestion, alveolar rupture, edema, and neutrophil infiltration. Serum levels of IL-6, IL-1β, VEGF, and ESM-1 were elevated, pulmonary expressions of F-actin, VE-cadherin, and ZO-1 were decreased, and H3Cit expression was increased significantly in the septic mice. GSK484 treatment effectively mitigated these changes in the septic mice. The LPS-stimulated endothelial cells showed increased productions of IL-6, IL-1β, VEGF and ESM-1, which were significantly reduced after treatment with 2.5 μmol/L GSK484.
Conclusions: GSK484 treatment effectively suppresses H3Cit expression in septic mice to ameliorate sepsis-induced endothelial dysfunction.
目的: 探讨PAD4抑制剂（GSK484）对脓毒症后H3Cit表达的抑制作用，并探究其对脓毒症引起的内皮功能障碍的改善效果。方法: C57BL/6小鼠18只通过盲肠结扎穿孔术（CLP）构建小鼠脓毒症模型，实验分为假手术组，模型组和GSK484治疗组，6只/组，治疗组在术后第2天腹腔注射给予GSK484（4 mg/kg）。ELISA检测小鼠血清VEGF、ESM-1、IL-6、IL-1β水平，HE染色观察肺组织病理学变化，免疫荧光和Western blotting检测各组小鼠肺组织中F-actin、VE-cadherin、ZO-1蛋白表达，Western blotting检测肺组织VE-Cadherin和H3Cit蛋白表达情况。肺组织原代分离培养肺微血管内皮细胞，使用脂多糖（LPS，10 μg/mL）干预24 h构建脓毒症模型。检测细胞成管能力，CCK-8检测细胞增殖，流式检测细胞凋亡，ELISA检测细胞上清中VEGF、ESM-1、IL-6、IL-1β水平。结果: 与假手术组相比，脓毒症小鼠肺组织肺脏血管充血、肺泡断裂、肺泡腔及肺间质水肿、肺泡隔增厚、中性粒细胞浸润等，血清中炎症因子IL-6、IL-1β和内皮细胞因子VEGF、ESM-1含量均升高（P<0.05），肺组织中胞间连接蛋白F-actin、VE-cadherin、ZO-1表达降低，H3Cit蛋白表达升高（P<0.05），GSK484治疗可以有效改善这些变化。LPS诱导肺微血管内皮细胞上清中炎症因子表达量升高、内皮细胞因子VEGF、ESM-1含量均升高（P<0.05），细胞成管能力降低；2.5 μmol/L浓度GSK484处理可降低炎症因子和内皮细胞因子表达（P<0.05）。结论: GSK484的使用可有效抑制脓毒症后H3Cit表达，进一步改善脓毒症后内皮功能障碍的发生。.
Keywords: GSK484; PAD4 inhibitor; endothelial dysfunction; lung injury; mouse pulmonary microvascular endothelial cells; sepsis.